The initiating event of many myocardial infarctions (heart attacks) is the hemorrhage into atherosclerotic plaque. Such hemorrhage often results in the formation of a thrombus (or blood clot) in the coronary artery which supplies the infarct zone (i.e., an area of coagulation necrosis which results from an obstruction of blood circulation). This thrombus is composed of a combination of fibrin and blood platelets. The formation of a fibrin-platelet clot has serious clinical ramifications. The degree and duration of the occlusion caused by the fibrin-platelet clot determines the mass of the infarct zone and the extent of damage.
The primary goal of current treatment for myocardial infarction involves the rapid dissolution of the occluding thrombus and the restoration of blood flow ("reperfusion"). A successful therapy must be capable of sustained effect so that reformation of the clot does not occur after the cessation of therapy. If the fibrin-platelet clot is able to reform, then the affected artery may become reoccluded.
The formation of fibrin-platelet clots in other parts of the circulatory system may be partially prevented through the use of anticoagulants (such as heparin). Unfortunately, heparin has not been found to be universally effective in preventing reocclusion in myocardial infarction victims in which the degree of blood vessel occlusion (the degree of "stenosis") is greater than or equal to 70%, particularly in those patients with severe residual coronary stenosis.
If an individual has formed a fibrin-platelet clot prior to the availability of medical assistance, the clot may be dissolved through the use of thrombolytic agents. A thrombolytic agent is a medicament capable of lysing the fibrin-platelet thrombus, and thereby permitting blood to again flow through the affected blood vessel. Such agents include streptokinase, prourokinase, urokinase, and tissue-type plasminogen activator (Ganz, W. et al., J. Amer. Coll. Cardiol. 1:1247-1253 (1983); Rentrop, K. P. et al., Amer. J. Cardiol. 54:29E-31E (1984); Gold, H. K. et al., Amer. J. Cardiol. 53:122C-125C (1984)).
Treatment with thrombolytic agents can often successfully restore coronary blood flow rapidly enough to interrupt myocardial infarction. Unfortunately, the dissolved fibrin-platelet clot has been found to reform after cessation of such thrombolytic therapy in a substantial number of patients. This reformation may result in the reocclusion of the affected blood vessels, and is, therefore, of substantial concern (Gold, H. K. et al., Amer. J. Cardiol. 53:122C-125C (1984); Gold, H. K. et al., Circulation 68:I-50-I-54 (1983)). Thus, although streptokinase treatment has been found to be successful in dissolving fibrin clots in approximately 85% of studied cases, reocclusion of the affected vessels has been found to occur in approximately 25% of the patients examined. (Gold, H. K., et al., Circulation 68:I50-I54 (1983)).
Tissue-type plasminogen activator (t-PA) is considered to be a more desirable thrombolytic agent than either streptokinase or urokinase because it displays greater (though not absolute) specificity for fibrin than does either of these agents (Verstrate, M., et al., Lancet 1:142 (1985)). Tissue-type plasminogen activator (t-PA) is a clot-specific thrombolytic agent with a rapid disposition rate from plasma. Tissue-type plasminogen activator (t-PA) has been found to be an effective thrombolytic agent in patients with acute myocardial infarction, producing coronary reflow (i.e., decreasing stenosis) in 45-75 minutes in approximately 70% of patients studied (Gold, H. K. et al., Circulation 73:347-352 (1986)).
The benefit of employing t-PA is significantly offset by the spontaneous rate of acute reocclusion which follows the cessation of t-PA therapy. Gold, H. K. and coworkers have found that cessation of t-PA therapy resulted in reocclusion of affected blood vessels in approximately 45% of patients studied (Circulation 73:347-352 (1986)). Increased t-PA dosages have not been found to decrease the tendency for coronary artery reocclusion. Significantly, the possibility of thrombin clot reformation is closely related to the degree of residual coronary stenosis (i.e., the extent of blood vessel blockage). Thus, reocclusion is more probable in individuals in which high grade stenosis (i.e., greater than 70% quantitative stenosis or greater than 80% non-quantitative stenosis) has occurred. The reocclusion of blood vessels has been found to be inhibited by continued infusion of t-PA (Gold, H. K. et al., Circulation 73:347-352 (1986)). Unfortunately, the relatively short biological half-life of t-PA and the potential for increasing the tendency for severe bleeding in some patients may make continued infusion of t-PA impractical for many heart attack victims.
In summary, clinical investigations have shown that the dissolved thrombus frequently reforms following the cessation of t-PA infusion (Gold, H. K. et al., Circulation 73:347-352 (1986)), but that the frequency of such reocclusion can be minimized by providing a second ("maintenance") t-PA infusion of a substantially lower dose but for a substantially longer period. Heparin is currently recognized as the appropriate concomitant therapy for patients receiving such a maintenance infusion. The treatment of coronary artery thrombosis (clotting) with t-PA requires, therefore, a continuous infusion at a high rate in order to obtain rapid reperfusion, and a maintenance infusion at a lower dose to prevent reocclusion in patients with high grade residual stenosis.
Clots are composed of both fibrin and blood platelets in various ratios. The fundamental reaction in blood clotting involves the conversion of a soluble plasma protein (fibrinogen) into insoluble fibrin. The conversion of fibrinogen into fibrin is catalyzed by the enzyme, thrombin, which is a serine protease. Fibrin molecules are then extensively crosslinked by Factor XIII to other fibrin molecules via their gamma and alpha chains. In addition, .alpha.-antiplasmin is crosslinked by Factor XIII to fibrin. Both of these crosslinking events result in a clot which is highly resistant to thrombolysis. The general mechanism of blood clot formation is reviewed by Ganong, W. F. (In: Review of Medical Physiology, 9th ed., Lange, Los Altos, Calif., pp. 411-414 (1979)). Platelets are disk-shaped structures present in blood which contribute to clot formation by both their incorporation along with fibrin into an insoluble mass and by providing additional Factor XIII, fibrin and .alpha.2-antiplasmin their enhancement of the rate of fibrinogen to fibrin conversion. Platelets contribute to clot formation in myocardial infarction and are a major component of clots that reocclude coronary arteries that have been reperfused by treatment with a thrombolytic agent.
Factor XIII, (fibrin stabilizing factor) in its active form, is responsible for the cross-linking of fibrin monomers in the final stages of the blood coagulation cascade. Factor XIII also crosslinks .alpha.2-antiplasmin to fibrin in plasma clots and thereby makes clots resistant to plasmin and plasminogen activators. The cross-linking of fibrin and cross-linking between fibrin, fibronectin and collagen appears to play a significant part in stabilizing the clot and promoting wound healing. Mosher et al. J. Biol. Chem. 255:1181-1188 (1980). Plasma factor XIII is a tetramer composed of two A subunits linked as a dimer and two loosely associated B subunits. Schwartz et al. J. Biol. Chem. 284:1395-1407 (1973). Plasma factor XIII crosslinks fibrin by introducing .gamma.-glutamyl-.epsilon.-lysyl bonds between the .gamma.-chains of neighboring units in the fibrin polymer and also between the chains of these subunits. Chen et al. Proc. Natl. Acad Sci. USA 66:472-479 (1970).
The A and B subunits of Factor XIII have been cloned and sequenced. Ichinose et al. Biochemistry 25:6900-6906 (1986); Gundman et al. Proc. Natl. Acad. Sci USA 83:8024-8028 (1986); and Ichinose et al. Biochemistry 25:4633-4638 (1986).
During coagulation the A subunit zymogen of Factor XIII is activated by the thrombin catalyzed cleavage of an amino terminal peptide (4 kDa). The activated A subunit catalyzes the formation of the peptide bonds between substrate polypeptides. The B subunit has no known catalytic activity and is known to protect the A subunit from rapid degradation.
Factor XIII is a proenzyme in the coagulation system which is located in plasmin, platelets and certain tissues. When activated in the terminal phase of coagulation, it functions as a transglutaminase in the terminal phase of coagulation and chemically crosslinks fibrin. Factor XIII activity is essential for normal hemostasis, and it also appears to play a critical role in thrombosis and in wound healing.
Clot lysis is mediated by plasmin in vivo. Under natural conditions, plasminogen is converted to plasmin by tissue plasminogen activator (t-PA). Activation occurs on the fibrin surface, thus confining proteolytic activity to the appropriate site. After plasmin is set free into the circulation, it is rapidly combined with natural inhibitors. Inactivation of plasmin is the final and necessary step in the process of protecting against undesirable proteolysis. Such plasmin inhibitors include .alpha.-2-antiplasmin, .alpha.-2-macroglobulin and .alpha.-1-antitrypsin, all glycoproteins. .alpha.-2-antiplasmin has a much higher affinity for plasmin than .alpha.-2-macroglobulin and binds specifically to plasmin in a 1:1 ratio. The larger pool of .alpha.-macroglobulin acts as a reservoir inhibitor. Kane, K. K., Ann. Clin. Lab. Sci. 14:443-449 (1984). Thus, clot lysis by the administration of t-PA is limited by the rapid and irreversible inactivation of plasmin by plasmin inhibitors.
All available thrombolytic agents still suffer significant shortcomings, including the need for large doses to be therapeutically efficient, a limited fibrin-specificity, residual toxicity in terms of bleeding complications by the fact that they may paradoxically activate platelets and increase clotting. Cardiovascular disease is still a major cause of disability. All current agents are associated with thrombotic reocclusion of blood vessels during or after therapy. Thus, there remains a need for additional agents which can be utilized alone or in combination with known thrombolytic agents. Improvements in thrombolytic therapy which enhance clot lysis, while minimizing fibrinogen breakdown and preventing reocclusion of the affected coronary artery are needed.